The proposed research is aimed at the development and application of an integrated microdialysis/mass spectrometry instrument technology for use in the in vivo analysis of the metabolism of neuropeptides and drugs in The instrument will utilize current quadrupole mass spectrometry (TSQ 70) equipped with a micro-electrospray ionization (micro-ESI) source, under development by the P.I., for very high sensitivity measurements, e.e., 1-5 femtomoles of compounds. The microdialysis probe is used as an in vivo tissue sampling device, with perfusion flows of about 500 nanoliters per minute of artificial cerebrospinal fluid. The micro-ESI source has been optimized to produce ions from the electrospray process at this flow rate. Proposed improvements of the micro-ESI source include lowering flow rates to 300 nanoliters/min, development of a computer-controlled automated sampling system for real-time measurements in vivo, and optimization of the source for high sensitivity. the application of combined microdialysis/micro-ESI mass spectrometry is described in four projects. i) Investigation of the kinetics and quantitative aspects of the in vivo metabolism of the tachykinin neuropeptides, including substance P, neurokinin a, neurokinin B, and neuropeptide K. and the enkephalin neuropeptides, including methionine-enkephalin, leucine-enkephalin, enkephalin heptapeptide, and enkephalin octapeptide. ii) Studies of the kinetics and differential processing of opioid peptide fragment precursors in vivo, including fragments of pro- opiomelanocortin, proenkephalin, and prodynorphin. The level of processing enzymes will also be measured. iii) Investigation of the in vivo quantitative production and enzymatic degradation of N-acetylaspartyl-glutamate in seizure disorders. iv) Development and measurements in vivo drug pharmacokinetics, using the drugs haloperidol, valproic acid, cocaine, amphetamine, and the new GABA antagonist n-butyl-3-aminopropylphosphinic acid. The significance of the techniques and applications proposed here is that they will be able to be applied directly to human health, especially in the area of critical care. It provides the clinicans and scientists a means to identify and monitor very low levels of natural compounds and drugs inmultiple sites directly and in real-time using a relatively non-invasive procedure. The mass specificity of the technology virtually eliminates ambiguities associated with other methodologies, such as radioimmunoassays, and the data can be obtained within 2-3 minutes of sampling.